MicroRNA (miRNA) are tiny posttranscriptional gene regulators, ˜20 nt oligoribonucleotides, that are differentially expressed during various diseases, such as heart failure and cancer, and have been implicated in the underlying pathogenesis. Each has the potential to regulate a set of specific genes that are involved in a common cellular function. For example, an array of growth-promoting genes are targeted by miR-1, and require its downregulation at the onset of cardiac hypertrophy. Since miRNA levels are posttranscriptionally regulated, they, therefore, have the potential to elicit an immediate and specific change in translation by attaching to, or detaching from, mRNA targets. Thus, an increase or a decrease in a specific miRNA may underlie the mechanism of these diseases.
Although mammalian miRNAs are commonly known for inhibiting translation vs. inducing mRNA degradation, there is now substantial evidence to support the latter as well. Therefore, it is plausible that transient exposure of an mRNA to a targeting miRNA will inhibit its translation while chronic exposure will result in its degradation.
Antisense miRNA is a critical tool for understanding the functions of the different miRNAs. Designing an expression vector of choice enhances the spectrum of our studies in the different cell lines and tissues as well as animal models. For example, cardiac myocytes are poor candidates for transfection and uptake of the cholesterol-linked oligos, in addition, to having a non-specific response to the cholesterol itself. On the other hand, they have great affinity to adenoviral vectors. The expression vectors can also be used to create transgenic mice models as a much faster alternative means for creating a knockout of a specific miRNA.
One approach to target a specific miRNA of interest has been to develop antisense sequences and deliver them to the cells via lipid based transfection methods or by attaching a cholesterol moiety to the oligonucleotide to render it cell permeable. The latter may be delivered in vivo with some success and has the potential to be used as a therapeutic agent. But like anything else this approach has its limitations and alternatives for different applications are always necessary.
This invention relates to an alternative strategy in which the antisense sequence of an miRNA of interest was expressed through an expression vector using a specific design that would allow for successful expression of 20-40 nucleotide sequences. This expression cassette can be delivered via plasmid DNA or viral vectors for more efficient in vivo and in vitro delivery. This system allows for continuous production of the antisense sequence and subsequently complete knockdown of the targeted miRNA.